In vitro tumor angiogenesis model

ABSTRACT

Provided is a method of inducing tubulogenesis in normal endothelial cells comprising co-culturing the normal endothelial cells with tumor cells and forming tubules from the normal endothelial cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/679,787, filed May 11, 2005, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an in vitro tumor angiogenesis model.The present invention further relates to a method of inducingtubulogenesis in normal endothelial cells.

BACKGROUND OF THE INVENTION

Endothelial cells that form the lining of blood vessels are well knownfor their capacity to adjust their numbers and arrangement to suitelocal requirements. All tissues depend on a blood supply and the bloodsupply depends on endothelial cells. Blood vessels create an adaptablelife support system in every region of the body. If not for endothelialcells extending and maintaining this network of blood vessels, tissuegrowth and repair would not be possible.

The largest blood vessels are arteries and veins, which have a thicktough outer wall of connective tissue and smooth muscle. The wall islined by a thin single layer of endothelial cells, separated from thesurrounding outer layers by a basal lamina. While the amounts ofconnective-tissue and smooth muscle in the vessel wall may varyaccording to the vessel's diameter and function, the endothelial liningis always present. In the smaller capillaries and sinusoids, the wallsconsist solely of endothelial cells and basal lamina. Thus, endothelialcells line the entire vascular system. Studies have shown that arteriesand veins develop from small vessels constructed solely of endothelialcells and a basal lamina, connective tissue and smooth muscle beingadded later where required upon signals from the endothelial cells.

Throughout the vascular system endothelial cells retain a capacity forcell division and movement. This is important in repair and maintenanceof the vascular system. For example, if a part of the wall of a bloodvessel is damaged and loses endothelial cells, neighboring endothelialcells will proliferate and migrate in to cover the exposed surface.Newly formed endothelial cells have also been known to cover the innersurface of plastic tubing used by surgeons to replace damaged bloodvessels.

Endothelial cells not only repair damaged blood vessels, they alsocreate new blood vessels. Endothelial cells do this in embryonic tissuesto support growth, in normal adult tissue for repair and maintenance,and in damaged tissue to support repair. This process is calledangiogenesis.

Angiogenesis is a critical component in embryonic development, tissuegrowth, tissue remodeling, and a number of pathologies. Angiogenesisresults in the formation of new blood vessels. During angiogenesis,endothelial cells, which exist in a quiescent state as part of anexisting blood vessel, grow and enter a migratory, proliferative state.This migratory, proliferative state is eventually resolved when thecells differentiate into capillary tubes and return to the quiescentstate as part of a functional new blood vessel. Angiogenesis isorchestrated by a complex network of multiple macromolecularinteractions.

Angiogenesis is regulated in both normal and malignant tissues by thebalance of angiogenic stimuli and angiogenic inhibitors that areproduced in the target tissue and at distant sites. Vascular endothelialgrowth factor-A (VEGF, also known as vascular permeability factor, VPF)is a primary stimulant of angiogenesis. VEGF is a multifunctionalcytokine that is induced by hypoxia and oncogenic mutations and can beproduced by a wide variety of tissues.

Angiogenesis is stimulated and harnessed by some neoplasms (e.g.,tumors) to increase nutrient uptake. However, in contrast to normalangiogenesis, which leads to anastomoses (i.e., vessel connections) andcapillary maturation, angiogenesis associated with neoplasia istypically a continuous process where vessel maturation is imperfect.Endothelial cells are activated by nearby neoplastic cells to secretenot only VEGF which stimulates angiogenesis, but also matrixmetalloproteases (MMP) which degrade the surrounding extracellularmatrix. The endothelial cells then invade the extracellular matrix wherethey proliferate, migrate, and organize to form new blood vessels, whichsupport neoplasm growth and survival.

The newly vascularized neoplasm continues to grow, leading to furthernutrient deprivation and chronic pro-angiogenic signaling. Thevasculature of neoplasms is characterized by the presence of structuralirregularities (lacunae) and a low rate of formation of inter-vesselconnections. This incomplete vasculature is inefficient, such that oftentumors require continuous angiogenesis to sustain themselves. Suchimperfect vasculature is also believed to promote the shedding ofneoplastic cells into the systemic circulation. Hence, the angiogenicpotential of a neoplasm correlates with metastatic potential. As asignificant proportion of neoplasms are dependent on continuedangiogenesis, inhibition of angiogenesis blocks neoplasm growth whichoften leads to complete necrosis of the neoplasm.

Glial cells including astrocytes comprise a large proportion of thetotal cell population in the central nervous system. Unlike neurons,glial cells retain the ability to proliferate postnatally, and someglial cells still proliferate in the adult or aged brain. Uncontrolledglial proliferation can lead to aggressive primary intracranial tumors.Such tumors vary widely in morphology and behavior, and, according tothe 1993 World Health Organization classification schema, can beseparated into three subsets. Astrocytomas, the lowest grade tumors, aregenerally well-differentiated and tend to grow slowly. Anaplasticastrocytomas are characterized by increased cellularity, nuclearpleomorphism (ability to assume different forms), and increased mitoticactivity. They are intermediate grade tumors and show a tendency toprogress to a more aggressive grade. Glioblastoma cells are consideredthe most aggressive, with poorly differentiated cells, vascularproliferation, and necrosis. Glioblastoma U251 is a malignant cell linederived from the human glial cells.

The angiogenic effects of glioblastoma cells and other solid tumor cellsin the presence of in vivo matrix effects and other in vivo ancillaryfactors, do not predict in vitro effectiveness in inducing primaryendothelial cells in culture to grow, and more particularly do notpredict in vitro effectiveness in inducing primary endothelial cells inculture to form tubules. The effectiveness of very low numbers of tumorcells to induce this effect is still more unexpected.

Non-normal, i.e., immortalized endothelial cells have been reported toform tubular structures in culture in the presence of glioblastomacells. But the growth of these kinds of cells can be expected to differconsiderably from normal cells, so the induction of tubules in normalcells could not be reliably predicted from this earlier work.

Thus, there is a need to induce angiogenic endothelial cells to betterenable collection of angiogenic endothelial cells for such purposes asangiogenic assay kits and in the study of endothelial cells,particularly the functions and permeability of the endothelial cellbarrier. Further, such cells have potential therapeutic uses

SUMMARY OF THE INVENTION

The present invention provides a method of inducing tubulogenesis innormal endothelial cells. The method includes co-culturing tumor cellswith normal endothelial cells and forming tubules from the normalendothelial cells. In some embodiments, the tumor cells may include, butare not limited to, glioblastoma cells, such as glioblastoma U251, orengineered or selected derivatives of glioblastoma cells (e.g.,transfected with VEGF). The normal endothelial cells are defined hereinas endothelial cells that have not been immortalized. These may include,but are not limited to, human dermal microvascular endothelial cells,human pulmonary microvascular endothelial cells or human umbilical veinendothelial cells.

In some embodiments, the step of co-culturing the tumor cells and normalendothelial cells includes incubating the endothelial cells and tumorcells in an endothelial cell culture medium for at least 1 day andforming tubules from the incubated endothelial cells.

The present invention further provides a method of preparing angiogenicnormal endothelial cells. The method includes co-culturing normalendothelial cells with tumor cells; and forming tubules from the normalendothelial cells. The method also includes selectively collecting thetubules, wherein the tubules include angiogenic normal endothelialcells. The “angiogenic normal endothelial cells” may be referred tointerchangeably herein as “selected normal endothelial cells”.

Further provided is a method of identifying bioactive agents. Theidentified bioactive agents may reduce or enhance tubulogenesis, forexample. The method includes co-culturing normal endothelial cells withtumor cells, thereby forming tubules from the normal endothelial cells.The method further includes contacting the normal endothelial cells witha prospective bioactive agent; and monitoring for changes in tubuleformation resulting from the presence of the bioactive agent. Thechanges are monitored relative to a control, i.e., the same methodconducted in the absence of the prospective bioactive agent.

DETAILED DESCRIPTION OF THE INVENTION

For simplicity and illustrative purposes, the principles of the presentinvention are described by referring to various exemplary embodimentsthereof. Although the preferred embodiments of the invention areparticularly disclosed herein, one of ordinary skill in the art willreadily recognize that the same principles are equally applicable to,and can be implicated in other compositions and methods, and that anysuch variation would be within such modifications that do not part fromthe scope of the present invention. Before explaining the disclosedembodiments of the present invention in detail, it is to be understoodthat the invention is not limited in its application to the details ofany particular embodiment shown, since of course the invention iscapable of other embodiments. The terminology used herein is for thepurpose of description and not of limitation. Further, although certainmethods are described with reference to certain steps that are presentedherein in certain order, in many instances, these steps may be performedin any order as may be appreciated by one skilled in the art, and themethods are not limited to the particular arrangement of steps disclosedherein.

Endothelial cells are the cells that make up the inside of bloodvessels. The term “normal endothelial cells” as used herein areendothelial cells that have not been immortalized. The term is meant toinclude human endothelial cells, as well as other mammalian and othervertebrate endothelial cells. Normal endothelial cells include, but arenot limited to, human dermal microvascular endothelial cells and humanpulmonary microvascular endothelial cells (HMVEC) and human umbilicalvein endothelial cells (HUVEC). These and other normal endothelial cellsare commercially available, for example, from BD Biosciences, a businesssegment of Becton, Dickinson & Co. (San Jose, Calif.), CascadeBiologies, Inc. (Portland, Oreg.) and Cambrex Corporation (EastRutherford, N.J.). Mammalian cells, particularly human cells, arepreferred. Selected normal endothelial cells comprise normal endothelialcells that are believed to have relatively high angiogenesis potential.These are alternatively referred to herein as angiogenic normalendothelial cells.

The method of inducing tubulogenesis involves co-culturing tumor cellswith normal endothelial cells. In a preferred embodiment, the methodincludes co-culturing tumor cells with endothelial cells, formingtubules from the normal endothelial cells and selectively collecting theformed tubules. The formed tubules include angiogenic normal endothelialcells. In one example, the tubules may be bound tentatively to thebottom surface of a well of a multi-well plate. The cell medium abovethe bound tubules in the well may include normal endothelial cells thathave not differentiated into tubules. Therefore, in some embodiments,the cell medium in the well is removed and the bound tubules are gentlywashed and then dislodged (e.g., mechanically) in a suitable volume offresh cell medium. In some embodiments, angiogenic endothelial cellsselectively collected in this way may be employed in angiogenesisassays, if desired.

In a preferred embodiment, the co-culturing is performed at anendothelial cell to tumor cell ratio of approximately 5:1 or more, or50:1 or more.

In an embodiment of the present invention, endothelial cells and tumorcells, such as glioblastoma U251, are harvested and then suspended in anendothelial cell culture medium. The desired number of each cell type isadded to a microwell and incubated. In a preferred embodiment, theincubation occurs for more than 1 day. In a more preferred embodiment,the incubation lasts for more than 2 days. In a preferred embodiment,incubation occurs in a 5% CO₂ incubator at 37 degrees Celsius.

Tumor cells that are suitable for use in the method are those that areknown to induce angiogenesis. For example, tumor cells known to induceangiogenesis include those from a wide variety of solid tumors, such asnon-small cell lung cancer, prostate cancer, breast cancer, gastriccancer, and tumors of the head and neck. Specific examples of angiogenictumor cell lines from these types of solid tumors are well known in theart, and may be obtained, for example, from American Type CultureCollection (ATCC, Manassas, Va.). In some preferred embodiments, thetumor cells are glioblastoma cells, such as glioblastoma U251. Ingeneral, suitable tumor cells release pro-angiogenic factors as signalsfor angiogenesis. Such pro-angiogenic factors include, for example,vascular endothelial growth factor (VEGF) acidic fibroblast growthfactor, angiogenin, basic fibroblast growth factor (bFGF), epidermalgrowth factor, granulocyte colony stimulating factor, hepactocyte growthfactor, interleukin-8, placental growth factor, platelet-derived growthfactor, endothelial growth factor, scatter factor, transforming growthfactor α and tumor necrosis factor α. Of these, two proteins appear tobe the most important for sustaining tumor growth: VEFG and bFGF. VEGFand bFGF are produced by many types of cancer cells and by certain typesof normal cells, too.

In some embodiments, tumor cell derivatives are employed in the presentinvention. For example, useful tumor cell derivatives include, but arenot limited to, those transfected with one or more of the pro-angiogenicfactors described above, particularly VEGF and/or bFGF. In someembodiments, the tumor cells are engineered or selected derivatives ofglioblastoma cells, such as glioblastoma U251 cells transfected withVEGF.

The method of inducing tubulogenesis can be incorporated into a methodof identifying bioactive agents by contacting prospective bioactiveagents with the endothelial cells at some time relevant to modulatingtubule formation. This time frame can be readily determinedexperimentally. Such time frame can be before the endothelial cells areco-cultured with tumor cells, or during the co-culturing. Bioactiveagents that reduce tubulogenesis are candidate bioactive agents fordisrupting blood vessel formation (angiogenesis) sought to be induced bytumor cells. Bioactive agents that stimulate tubulogenesis are candidatebioactive agents to increase vascularization in tissue damaged byischemic events, in tissue whose vascularization has been damaged byenvironmental factors such as smoking, and in and aged-relatedblindness. Tubulogenesis can be most simply monitored visually. However,tubule formation can be assessed quantitatively, if desired. Forexample, following tubulogenesis, the assays may be stained with CalceinAM, and images may be acquired and total tube length measured using animage analyzer (e.g., using MetaMorph from Universal ImagingCorporation). Tubulogenesis can also be monitored by assaying the levelsof molecules that function as surrogate markers for tubulogenesis. Suchmethods of monitoring tubulogenesis are known in the art.

A bioactive agent is a substance that can act on a cell, tissue, organor organism to create a change in the functioning of the cell, organ ororganism. The prospective bioactive agent may be a chemical, such as apharmaceutical agent. Other prospective bioactive agents include, butare not limited to, insecticides, herbicides, proteins, (e.g., growthfactors and cytokines), peptides, etc.

A tubule formation altering effective amount of a bioactive agent is anamount effective to change the rate (to faster or slower), degree(greater or lesser) or morphology of tubule formation, or to change(upwards or downwards) another measure of tubulogenesis. The change ismeasured relative to a control tubule formation assay where thebioactive agent is not present.

EXAMPLE

Two types of primary human endothelial cells, HUVEC and HMVEC were eachmixed at a ratio of 100:1 with tumor cell U251. The cells were seeded in6-well plates at a density of 1×10⁶/well. The co-cultures were incubatedin endothelial cell medium EGM-2 MV (Cambrex, Walkersville, Md.) in a37° C./5% CO₂ incubator and the medium was replaced every two days untilday 11. Tube formation was visually observed on and after day 7 in bothco-cultures.

Alternatively, tube formation is assessed after at least one day ofincubating the co-cultures at 37° C./5% CO₂ by staining with Calcein AM.Images are thereafter acquired using a 2× objective lens and total tubelength is measured using MetaMorph (Universal Imaging Corporation).

As described above, in some embodiments, the assays described in thepresent example are used to identify bioactive agents that inhibit orstimulate tubulogenesis. For example, normal endothelial cells, such asHUVEC and HMVEC, may be contacted with different amounts of aprospective bioactive agent before the normal endothelial cells areco-cultured with the tumor cells, and changes in tubule formationresulting from the presence of the bioactive agent may be monitored.Alternatively, different amounts of a prospective bioactive agent may becontacted with the normal endothelial cells during the co-culturingstep, and changes in the tubule formation resulting from the presence ofthe bioactive agent may be monitored.

While the invention has been described with an emphasis on particularembodiments thereof, those skilled in the art may make variousmodifications to the described embodiments of the invention withoutdeparting from the scope of the invention. Although the invention hasbeen described and disclosed in various terms and certain embodiments,the scope of the invention is not intended to be, nor should it bedeemed to be, limited thereby and such other modifications orembodiments as may be suggested by the teachings herein are particularlyreserved, especially as they fall within the breadth and scope of theclaims here appended. Those skilled in the art will recognize that theseand other variations are possible within the scope of the invention asdefined in the following claims and their equivalents.

1. An in vitro method of inducing tubulogenesis in normal endothelialcells comprising mixing and co-culturing the normal endothelial cellswith tumor cells and forming tubules from the normal endothelial cells,wherein the normal endothelial cells are human endothelial cells thathave not been immortalized and wherein the tumor cells compriseglioblastoma cells and the ratio of normal endothelial cells to tumorcells is at least 5:1.
 2. The method of claim 1, wherein theglioblastoma cells comprise glioblastoma U251.
 3. The method of claim 1,wherein the normal endothelial cells are selected from the groupconsisting of human dermal microvascular endothelial cells, humanpulmonary microvascular endothelial cells and human umbilical veinendothelial cells.
 4. The method of claim 1, wherein the ratio of normalendothelial cells to tumor cells is at least 50:1.
 5. The method ofclaim 1, wherein the step of co-culturing comprises: incubating thenormal endothelial cells and tumor cells in an endothelial cell culturemedium for at least 1 day; and forming tubules from the incubatedendothelial cells.
 6. The method of claim 5, wherein the step ofincubating the normal endothelial cells and tumor cells is performed forat least 2 days.
 7. The method of claim 5, wherein the step ofincubating the normal endothelial cells and tumor cells occurs in a 5%CO₂ incubator at 37 degrees Celsius.